Alteration of sequence of a target molecule

ABSTRACT

Method for splicing a target nucleic acid molecule with a separate nucleic acid molecule. Such splicing generally causes production of a chimeric protein with advantageous features over that protein naturally produced from the target nucleic acid prior to splicing. The method includes contacting the target nucleic acid molecule with a catalytic nucleic acid molecule including the separate nucleic acid molecule. Such contacting is performed under conditions in which at least a portion of the separate nucleic acid molecule is spliced with at least a portion of the target nucleic acid molecule to form a chimeric nucleic acid molecule. In this method, the catalytic nucleic molecule is chosen so that it is not naturally associated with the separate nucleic acid molecule.

This application is a CON of Ser. No. 08/786,753 Jan. 24, 1997 U.S. Pat.No. 5,869,254 which is a CON of Ser. No. 08/152,450 Nov. 12, 1993 U.S.Pat. No. 5,667,969.

This invention relates to therapy of diseases using ribozymes.

The following is a brief history of the discovery and activity ofenzymatic RNA molecules or ribozymes. This history is not meant to becomplete but is provided only for understanding of the invention thatfollows. This summary is not an admission that all of the work describedbelow is prior art to the claimed invention.

Prior to the 1970s it was thought that all genes were direct linearrepresentations of the proteins that they encoded. This simplistic viewimplied that all genes were like ticker tape messages, with each tripletof DNA “letters” representing one protein “word” in the translation.Protein synthesis occurred by first transcribing a gene from DNA intoRNA (letter for letter) and then translating the RNA into protein (threeletters at a time). In the mid 1970s it was discovered that some geneswere not exact, linear representations of the proteins that they encode.These genes were found to contain interruptions in the coding sequencewhich were removed from, or “spliced out” of, the RNA before it becametranslated into protein. These interruptions in the coding sequence weregiven the name of intervening sequences (or introns) and the process ofremoving them from the RNA was termed splicing. A general reference forspliceosomes and how they are related to self-splicing introns isGuthrie, C., 253 Science 157, 1991. After the discovery of introns, twoquestions immediately arose: (i) why are introns present in genes in thefirst place, and (ii) how do they get removed from the RNA prior toprotein synthesis? The first question is still being debated, with noclear answer yet available. The second question, how introns get removedfrom the RNA, is much better understood after a decade and a half ofintense research on this question. At least three different mechanismshave been discovered for removing introns from RNA. Two of thesesplicing mechanisms involve the binding of multiple protein factorswhich then act to correctly cut and join the RNA. A third mechanisminvolves cutting and joining of the RNA by the intron itself, in whatwas the first discovery of catalytic RNA molecules.

Cech and colleagues were trying to understand how RNA splicing wasaccomplished in a single-celled pond organism called Tetrahymenathermophila. They had chosen Tetrahymena thermophila as a matter ofconvenience, since each individual cell contains over 10,000 copies ofone intron-containing gene (the gene for ribosomal RNA). They reasonedthat such a large number of intron-containing RNA molecules wouldrequire a large amount of (protein) splicing factors to get the intronsremoved quickly. Their goal was to purify these hypothesized splicingfactors and to demonstrate that the purified factors could splice theintron-containing RNA in vitro. Cech rapidly succeeded in getting RNAsplicing to work in vitro, but something funny was going on. Asexpected, splicing occurred when the intron-containing RNA was mixedwith protein-containing extracts from Tetrahymena, but splicing alsooccurred when the protein extracts were left out. Cech proved that theintervening sequence RNA was acting as its own splicing factor to snipitself out of the surrounding RNA. They published this startlingdiscovery in 1982. Continuing studies in the early 1980's served toelucidate the complicated structure of the Tetrahymena intron and todecipher the mechanism by which self-splicing occurs. Many researchgroups helped to demonstrate that the specific folding of theTetrahymena intron is critical for bringing together the parts of theRNA that will be cut and spliced. Even after splicing is complete thereleased intron maintains its catalytic structure. As a consequence, thereleased intron is capable of carrying out additional cleavage andsplicing reactions on itself (to form intron circles). By 1986, Cech wasable to show that a shortened form of the Tetrahymena intron could carryout a variety of cutting and joining reactions on other pieces of RNA.The demonstration proved that the Tetrahymena intron can act as a trueenzyme: (i) each intron molecule was able to cut many substratemolecules while the intron molecule remained unchanged, and (ii)reactions were specific for RNA molecules that contained a uniquesequence (CUCU) which allowed the intron to recognize and bind the RNA.Zaug and Cech coined the term “ribozyme” to describe any ribonucleicacid molecule that has enzyme-like properties. Also in 1986, Cech showedthat the RNA substrate sequence recognized by the Tetrahymena ribozymecould be changed by altering a sequence within the ribozyme itself. Thisproperty has led to the development of a number of site-specificribozymes that have been individually designed to cleave at other RNAsequences. The Tetrahymena intron is the most well-studied of what isnow recognized as a large class of introns, Group I introns. The overallfolded structure, including several sequence elements, is conservedamong the Group I introns, as is the general mechanism of splicing. Likethe Tetrahymena intron, some members of this class are catalytic, i.e.the intron itself is capable of the self-splicing reaction. Other GroupI introns require additional (protein) factors, presumably to help theintron fold into and/or maintain its active structure. While theTetrahymena intron is relatively large, (413 nucleotides) a shortenedform of at least one other catalytic intron (SunY intron of phage T4,180 nucleotides) may prove advantageous not only because of its smallersize but because it undergoes self-splicing at an even faster rate thanthe Tetrahymena intron.

Ribonuclease P (RNAseP) is an enzyme comprised of both RNA and proteincomponents which are responsible for converting precursor tRNA moleculesinto their final form by trimming extra RNA off one of their ends.RNAseP activity has been found in all organisms tested, but thebacterial, enzymes have been the most studied. The function of RNAsePhas been studied since the mid-1970s by many labs. In the late 1970s,Sidney Altman and his colleagues showed that the RNA component of RNAsePis essential for its processing activity; however, they also showed thatthe protein component also was required for processing under theirexperimental conditions. After Cech's discovery of self-splicing by theTetrahymena intron, the requirement for both protein and RNA componentsin RNAseP was reexamined. In 1983, Altman and Pace showed that the RNAwas the enzymatic component of the RNAseP complex. This demonstratedthat an RNA molecule was capable of acting as a true enzyme, processingnumerous tRNA molecules without itself undergoing any change. The foldedstructure of RNAseP RNA has been determined, and while the sequence isnot strictly conserved between RNAs from different organisms, thishigher order structure is. It is thought that the protein component ofthe RNAseP complex may serve to stabilize the folded RNA in vivo. Atleast one RNA position important both to substrate recognition and todetermination of the cleavage site has been identified, however littleelse is known about the active site. Because tRNA sequence recognitionis minimal, it is clear that some aspect(s) of the tRNA structure mustalso be involved in substrate recognition and cleavage activity. Thesize of RNAseP RNA (>350 nucleotides), and the complexity of thesubstrate recognition, may limit the potential for the use of anRNAseP-like RNA in therapeutics. However, the size of RNAseP is beingtrimmed down (a molecule of only 290 nucleotides functions reasonablywell). In addition, substrate recognition has been simplified by therecent discovery that RNAseP RNA an cleave small RNAs lacking thenatural tRNA secondary structure if an additional RNA (containing a“guide” sequence and a sequence element naturally present at the end ofall tRNAs) is present as well.

Symons and colleagues identified two examples of a self-cleaving RNAthat differed from other forms of catalytic RNA already reported. Symonswas studying the propagation of the avocado sunblotch viroid (ASV), anRNA virus that infects avocado plants. Symons demonstrated that aslittle as 55 nucleotides of the ASV RNA was capable of folding in such away as to cut itself into two pieces. It is thought that in vivoself-cleavage of these RNAs is responsible for cutting the RNA intosingle genome-length pieces during viral propagation. Symons discoveredthat variations on the minimal catalytic sequence from ASV could befound in a number of other plant pathogenic RNAs as well. Comparison ofthese sequences revealed a common structural design consisting of threestems and loops connected by central loop containing many conserved(invariant from one RNA to the next) nucleotides. The predictedsecondary structure for this catalytic RNA reminded the researchers ofthe head of a hammer; thus it was named as such. Uhlenbeck wassuccessful in separating the catalytic region of the ribozyme from thatof the substrate. Thus, it became possible to assemble a hammerheadribozyme from 2 (or 3) small synthetic RNAs. A 19-nucleotide catalyticregion and a 24-nucleotide substrate were sufficient to support specificcleavage. The catalytic domain of numerous hammerhead ribozymes have nowbeen studied by both the Uhlenbeck and Symons groups with regard todefining the nucleotides required for specific assembly and catalyticactivity and determining the rates of cleavage under various conditions.

Haseloff and Gerlach showed it was possible to divide the domains of thehammerhead ribozyme in a different manner. By doing so, they placed mostof the required sequences in the strand that didn't get cut (theribozyme) and only a required UH where H=C, A, or U in the strand thatdid get cut (the substrate). This resulted in a catalytic ribozyme thatcould be designed to cleave any UH RNA sequence embedded within a longer“substrate recognition” sequence. The specific cleavage of a long mRNA,in a predictable manner using several such hammerhead ribozymes, wasreported in 1988.

One plant pathogen RNA (from the negative strand of the tobacco ringspotvirus) undergoes self-cleavage but cannot be folded into the consensushammerhead structure described above. Bruening and colleagues haveindependently identified a 50-nucleotide catalytic domain for this RNA.In 1990, Hampel and Tritz succeeded in dividing the catalytic domaininto two parts that could act as substrate and ribozyme in amultiple-turnover, cutting reaction. As with the hammerhead ribozyme,the hairpin catalytic portion contains most of the sequences requiredfor catalytic activity while only a short sequence (GUC in this case) isrequired in the target. Hampel and Tritz described the folded structureof this RNA as consisting of a single hairpin and coined the term“hairpin” ribozyme (Bruening and colleagues use the term “paper clip”for this ribozyme motif). Continuing experiments suggest an increasingnumber of similarities between the hairpin and hammerhead ribozymes inrespect to both binding of target RNA and mechanism of cleavage. At thesame time, the minimal size of the hairpin ribozyme is still 50-60%larger than the minimal hammerhead ribozyme.

Hepatitis Delta Virus (HDV) is a virus whose genome consists ofsingle-stranded RNA. A small region (−80 nucleotides) in both thegenomic RNA, and in the complementary anti-genomic RNA, is sufficient tosupport self-cleavage. As the most recently discovered ribozyme, HDV'sability to self-cleave has only been studied for a few years, but isinteresting because of its connection to human disease. In 1991, Beenand Perrotta proposed a secondary structure for the HDV RNAs that isconserved between the genomic and anti-genomic RNAs and is necessary forcatalytic activity. Separation of the HDV RNA into “ribozyme” and“substrate” portions has recently been achieved by Been, but the rulesfor targeting different substrate RNAs have not yet been determinedfully. Been has also succeeded in reducing the size of the HDV ribozymeto −60 nucleotides.

The table below lists some of the characteristics of the ribozymesdiscussed above:

TABLE 1 Characteristics of ribozymes Group I Introns Size: ˜300 to >1000nucleotides. Requires a U in the target sequence immediately 5′ of thecleavage site. Binds 4-6 nucleotides at 5′ side of cleavage site. Over75 known members of this class. Found in Tetrahymena thermophila rRNA,fungal mitochondria, chloroplasts, phage T4, blue-green algae, andothers. RNAseP RNA (M1 RNA) Size: ˜290 to 400 nucleotides. RNA portionof a ribonucleoprotein enzyme. Cleaves tRNA precursors to form maturetRNA. Roughly 10 known members of this group all are bacterial inorigin. Hammerhead Ribozyme Size: ˜30 to 40 nucleotides. Requires thetarget sequence UH immediately 5′ of the cleavage site. Binds a variablenumber nucleotides on both sides of the cleavage site. 14 known membersof this class. Found in a number of plant pathogens (virusoids) that useRNA as the infectious agent. Hairpin Ribozyme Size: ˜50 nucleotides.Requires the target sequence GUC immediately 3′ of the cleavage site.Binds 4 nucleotides at 5′ side of the cleavage site and a variablenumber to the 3′ side of the cleavage site. Only 1 known member of thisclass. Found in one plant pathogen (satellite RNA of the tobaccoringspot virus) which uses RNA as the infectious agent. Hepatitis DeltaVirus (HDV) Ribozyme Size: ˜60 nucleotides (at present). Cleavage oftarget RNAs recently demonstrated. Sequence requirements not fullydetermined. Binding sites and structural requirements not fullydetermined, although no sequences 5′ of cleavage site are required. Only1 known member of this class. Found in human HDV.

As the term is used in this application, ribozymes are RNA moleculeshaving an enzymatic activity which is able to cleave and splice otherseparate RNA molecules in a nucleotide base sequence specific manner.Such enzymatic RNA molecules can be targeted to virtually any RNAtranscript, and efficient cleavage and splicing achieved in vitro. Kimet al., 84 Proc. Nat. Acad. of Sci. USA 8788, 1987, Hazeloff et al., 234Nature 585, 1988, Cech, 260 JAMA 3030, 1988, and Jefferies et al., 17Nucleic Acid Research 1371, 1989.

Ribozymes act by first binding to a target RNA. Such binding occursthrough the target RNA binding portion of a ribozyme which is held inclose proximity to an enzymatic portion of the RNA which acts to cleavethe target RNA. Thus, the ribozyme first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts to cut and splice the target RNA. Strategic cleavageand splicing of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After a ribozyme has bound, cleaved andspliced its RNA target it is released from that RNA.

By the phrase “catalytic” or “enzymatic RNA molecule” is meant an RNAmolecule which has complementarity in a substrate binding region to aspecified gene target, and also has an enzymatic activity which isactive to specifically cleave and splice RNA in that target. That is,the enzymatic RNA molecule is able to intermolecularly cleave and spliceRNA and thereby alter a target RNA molecule. This complementarityfunctions to allow sufficient hybridization of the enzymatic RNAmolecule to the target RNA to allow the cleavage to occur. 100%complementarity is preferred, but complementarity as low as 50-75% mayalso be useful in this invention.

In preferred embodiments of this invention, the enzymatic RNA moleculeis formed in a hammerhead motif, but may also be formed in the motif ofa hairpin, hepatitis delta virus, group I intron or RNAseP RNA (inassociation with an RNA guide sequence). Examples of such hammerheadmotifs are described by Rossi et al., 8 AIDS RESEARCH AND HUMANRETROVIRUSES 183, 1992, of hairpin motifs by Hampel et al., RNA CATALYSTFOR CLEAVING SPECIFIC RNA SEQUENCES, filed Sep. 20, 1989, which is acontinuation-in-part of U.S. Ser. No. 07/247,100 filed Sep. 20, 1988,Hampel and Tritz, 28 Biochemistry 4929, 1989 and Hampel et al., 18Nucleic Acids Research 299, 1990, and an example of the hepatitis deltavirus motif is described by Perrotta and Been, 31 Biochemistry 16, 1992,of the RNAseP motif by Guerrier-Takada at al., 35 Cell 849, 1983, and ofthe group I intron by Cech et al., U.S. Pat. No. 4,987,071. Thesespecific motifs are not limiting in the invention and those skilled inthe art will recognize that all that is important in an enzymatic RNAmolecule of this invention is that it has a specific substrate bindingsite which is complementary to one or more of the target gene RNAregions, and that it have nucleotide sequences within or surroundingthat substrate binding site which impart an RNA cleaving activity to themolecule.

The invention provides a method for designing a class of enzymaticcleaving and splicing agents which exhibit a high degree of specificityfor the RNA of a desired target. The ribozyme molecule is preferablytargeted to a highly conserved sequence region of a target such thatspecific treatment of a disease or condition can be provided with asingle ribozyme. Such enzymatic RNA molecules can be deliveredexogenously to specific cells as required.

Synthesis of ribozymes greater than 100 nucleotides in length is verydifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. However, delivery of such ribozymes byexpression vectors is primarily feasible using ex vivo treatments.

Inoue et al., 43 Cell 431, 1985, state that short oligonucleotides of2-6 nucleotides can undergo intermolecular exon ligation or splicing intrans. It indicates that “long 5′ exons should be reactive provided thatthree conditions are met: the exon must have a 3′ hydroxyl group, itmust terminate in a sequence similar to that of the 3′ end of the 5′exon, and the 3′ terminal sequence must be available as opposed to beingtied up in some secondary structure. Thus, it appears that exonswitching is possible in this system, though limited by the availabilityof alternative 5′ exons that meet the above criteria. These couldinclude transcripts that are not 5′ exons from other precursors, sinceRNA polymerases always leave 3′ hydroxyl ends”.

SUMMARY OF THE INVENTION

This invention features a method in which natural transcripts arealtered by use of a splicing reaction in vivo or in vitro. It involvesthe manipulation of genetic information to ensure that a usefultranscript is provided within a cellular system or extract.

In a first aspect, the invention features a method for splicing a targetnucleic acid molecule with a separate nucleic acid molecule. Suchsplicing generally causes production of a chimeric protein withadvantageous features over that protein naturally produced from thetarget nucleic acid prior to splicing. The method includes contactingthe target nucleic acid molecule with a catalytic nucleic acid moleculeincluding the separate nucleic acid molecule. Such contacting isperformed under conditions in which at least a portion of the separatenucleic acid molecule is spliced with at least a portion of the targetnucleic acid molecule to form a chimeric nucleic acid molecule. In thismethod, the catalytic nucleic acid molecule is chosen so that it is notnaturally associated with the separate nucleic acid molecule.

The target nucleic acid molecule can be any desired molecule with whicha splicing reaction can occur. Generally, this will be an RNA molecule,preferably a messenger RNA molecule, but it may also include moleculesthat have one or more non-ribonucleotides substituents, such asdeoxyribonucleotides or other analogs as described by Usman et al.,PCT/US93/00833 and Eckstein et al. EP90/01731, both hereby incorporatedby reference.

Generally, the target nucleic acid molecule is present within a cell andis chosen or targeted because it encodes a defective protein or isdeleterious to that cell. Splicing of the separate nucleic acid moleculewith such a target nucleic molecule is designed to alter the proteinproduct of that nucleic acid molecule. Such alteration causes productionof a useful protein which will allow that cell to either survive or die,as desired. Thus, for example, in a gene therapy setting, the targetnucleic acid molecule may encode a non-functional protein necessary fornormal life. This molecule can be spliced with a separate nucleic acidmolecule to allow appropriate expression of a functional protein.Alternatively, the splicing may cause production of a more stableprotein, or of a protein which acts as an agonist or antagonist of afunction, e.g., a viral or bacterial replication function.

The separate nucleic acid molecule is generally chosen such that itencodes a 3′ axon which it is desirable to express within a cell. Thisexon will generally not include control sequences such as promoterregions, but may include poly(A) tails and other stabilizing orenhancing functions well known in the art. As with the target nucleicacid molecule, the separate nucleic acid molecule generally is aribonucleic acid molecule but may be substituted as described above.

By “enzymatic” or “catalytic nucleic acid molecule” is meant a moleculehaving a motif generally as described above in the Background of theInvention, which and is preferably selected from the motif of a group Ior group II intron having a cleavage and splicing activity.Alternatively, the splicing or cleavage activity may be provided by adifferent nucleic acid molecule, or may supplement the catalytic nucleicacid molecule. Those of ordinary skill in the art will recognize thatother motifs than those of the group I and group II introns may also bemanipulated to provide useful splicing activity.

The conditions chosen for the contacting step may be those naturallyoccurring within a cell, or may be manipulated in vitro to ensure thatthe splicing reaction will occur. These conditions are well known tothose in the art, for example, as described by Inoue et al., supra.

By at least a portion of the respective nucleic acid molecules is meantthat the 5′ end of the target nucleic acid molecule will be spliced withthe 3′ end of the separate nucleic acid molecule. Such a portion may beonly a few nucleotides (10-500 nucleotides) or may be significantlygreater and may represent almost all of a molecule encoding a geneproduct (i.e., at least 1 to 5 kbases).

The chimeric nucleic acid molecule is one which may occur naturally innature but is not present prior to the splicing reaction. Alternatively,it may be a completely novel structure which does not occur in nature,but which is useful in gene therapeutic treatment of an organism.

The catalytic nucleic acid molecule is not naturally associated with theseparate nucleic acid molecule since it is not generally desired tosplice the 3′ end of a naturally occurring catalytic nucleic acidmolecule with a target nucleic acid molecule. Rather, the separatenucleic acid molecule is chosen or selected to have a beneficialfunction once spliced with the target nucleic molecule.

In a related aspect, the invention features a method for splicing atarget nucleic acid molecule with a separate nucleic acid molecule bycontacting those molecules in the presence of one or more splicingfactors or spliceosomes under splicing conditions. Such molecules arenot naturally spliced together in nature, although the final spliceproduct may be a natural product.

The various splicing factors and spliceosomes are well known in the art,and this activity is generally described by Bruzik and Maniatis in 360Nature 692, 1992, hereby incorporated by reference herein. The inventionconcerns splicing of target nucleic acid molecules and separate nucleicacid molecules which are not normally spliced together within a cell asdescribed by Bruzik and Maniatis, supra. Rather, as described above, aseparate nucleic acid molecule is selected such that a useful functioncan be achieved in a gene therapeutic fashion.

In preferred embodiments, the catalytic nucleic acid is able to cleaveand splice, e.g., it has a group I or group II intron motif; the methodis performed in vitro or in vivo with an RNA target; and the method canbe used to treat genetic disease in a gene therapy type manner, forexample, by correcting an abnormal transcript, or by providing antiviralactivity such as a dominant negative allele to a viral RNA.

In other aspects, the invention features catalytic nucleic acidmolecules having a selected separate nucleic acid molecule as a 3′ exonencoding at least a portion of a useful gene which can be used in genetherapy. Such a molecule can be spliced with and thereby correct ormodify the expression of other target RNA molecules. The invention alsofeatures vectors encoding such catalytic nucleic acid molecules.

The observation that ribozymes can specifically cleave targeted RNAs invitro has led to much speculation about their potential usefulness asgene inhibitors. By cleaving targeted mRNAs in vivo, ribozymes can beused to stop the flow of genetic information. Here we describe adifferent application of ribozymes. For example, a group I intronribozyme can be used to manipulate the flow of genetic information bytargeted trans-splicing. Defective cellular transcripts may be repaired,or pathogen-derived transcripts may be altered to encode antagonists tothe pathogen using such technology.

In nature the group I intron ribozyme from Tetrahymena thermophilaself-splices itself from precursor ribosomal RNAs (T. R. Cech, A. J.Zaug, P. J. Grabowski, Cell 27 487 (1981); K. Kruger et al., Cell 31,147 (1982)). This process is accomplished in two successive steps. Firstthe phosphodiester bond at the 5′ exon-intron border is cleaved. Thenthe 3′ hydroxyl group on the 5′ exon is covalently attached to the 3′exon, and the intron is removed (FIG. 1A). It has been previouslydemonstrated in vitro that the 5′ exon in this reaction can be mimickedby RNA molecules supplied in trans; the minimum active unit is thedinucleotide substrate rCU (FIG. 1B) (T. Inoue, F. X. Sullivan, T. R.Cech, Cell 43, 431 (1985)). We propose use of trans-splicing reactionsto ligate foreign sequences onto targeted transcripts after cleavage(FIG. 1C). In this manner, ribozymes can be employed to manipulate theflow of genetic information inside cells by changing what a targeted RNAencodes.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The drawings will briefly be described.

Drawings

FIGS. 1A, 1B, and 1C are diagrammatic representations showing reactionsof the group I intron from Tetrahymena for targeted trans-splicing.

FIGS. 2K and 2B are a comparison of cis- and trans-splicing reactionsfor LacZ transcripts.

FIG. 3 is a copy of an autoradiogram showing targeted trans-splicing tocorrect truncated transcripts from the alpha complement of LacZ 39nucleotides long. L-21 (or L-21 del) ribozyme-3′ exon chimeric RNAs (seeFIG. 2B) (³²P-body-labeled) (200 nM) were preheated in reaction buffer[50 mM Hepes (pH 7.0), 150 mM NaCl, and 5 mM MgCl₂] at 50° C. for 5minutes and then equilibrated at 37° C. for 2 minutes. The 13 (5′-A₅:GGCCCUCUA₅) or 39 (5′L-A₂: see FIG. 2B) nucleotide substrate RNAs (1 μM)and GTP (100 μM) were preheated to 37° C. and added to the ribozymes tostart the reactions which proceeded at 37° C. Portions containing onefifteenth of the reactions were removed at 0, 2, 10, 60, and 180 minutesand added to an equal volume of 10 mM EDTA to stop the reactions.Reaction products were analyzed upon a 4% polyacrylamide gel with 8 Murea. The inactive L-21 del ribozyme was generated by deleting 93nucleotide of the ribozyme (nucleotides 237-330 comprising L6b to P9).

FIG. 4 is a graphical representation of the targeted trans-splicing ratefor correcting the 39 nucleotide truncated LacZ transcript. The productsfrom the trans-splicing reaction time course containing the action L-21ribozyme and the 39 nucleotide substrate shown in FIG. 2 were quantifiedwith an AMBIS Image Acquisition and Analysis System (AMBIS, Inc., SanDiego, Calif.). The percentage of the ribozyme-3′ exon RNA remaining isplotted versus time.

FIG. 5 is a copy of an autoradiogram showing hydrolysis of the 3′ LacZexon attached to the L-21 ribozyme. L-21 (or L-21 del) ribozyme-3′ exonchimeric RNAs (³²P-body-labeled) (100 nM) wee incubated at 37° C. inreaction buffer [50 mM Hepes (pH 7.0), 150 mM NaCl, 5 mM MgCl₂, and 100μM GTP]. A portion of the reaction was removed after 0, 2, 10, and 60minutes and added to an equal volume of 10 mM EDTA to stop the reaction.Products were analyzed upon a 4% polyacrylamide gel containing 8 M urea.

FIGS. 6A and 6B are representations of trans-splicing to recreate anentire 3074 nucleotide LacZ messenger RNA from a 1106 nucleotidetruncated transcript. A. Scheme for correcting transcript. B.Trans-splicing reaction. L-21 (or L-21 del) ribozyme-3′ exon chimericRNAs (20 nM) were preheated in reaction buffer [50 mM Hepes (pH 7.0),150 mM NaCl, and 5 mM MgCl₂] at 50° C. for 5 minutes and thenequilibrated at 37° C. for 2 minutes. The 1106 nucleotide substrate RNA(³²P-end labeled) (200 nM) and GTP (100 μM) were preheated at 37° C. andadded to the ribozymes to start the reactions which proceeded at 37° C.One sixth of each reaction was removed at 0, 2, 10, 60, and 180 minutesand added to an equal volume of 10 mM EDTA to stop the reaction.Reaction products were analyzed upon a 1.2% agarose gel containing 1.1%formaldehyde. rRNAs from mouse NIH 3T3 cells were used to as 5100 and1900 nt molecular weight markers. The remaining sixth of the reactions,which had proceeded for 120 minutes, were in vitro translated.

FIG. 7 is a scheme for correcting genetic mutations using targetedtrans-splicing.

FIG. 8 is a scheme for mutating HIV transcripts using targetedtrans-splicing.

Targeted Trans-Splicing

The general scheme for a targeted trans-splicing is shown in FIG. 1using the group I intron of Tetrahymena thermophila as an example. Thosein the art will recognize that this example is not limiting in theinvention and that other enzymatic RNA molecules having the appropriatesplicing activity can be used in the invention. Alternatively, asdiscussed above, these molecules can be supplemented by other moleculeshaving a suitable splicing activity, or by spliceosomes or splicingfactors. Generally, the reaction involves base pairing of the catalyticnucleic acid molecule with the targeted transcript, cleavage of thetargeted transcript, and then ligation of the 3′ exon (separate nucleicacid molecule) with this targeted 5′ exon. The catalytic nucleic acid isremoved in the reaction. As will be noted, the specificity of thereaction can be changed by alteration of the substrate binding site inthe catalytic nucleic acid molecule by methods well known in the art.

The following is an example of various constructs used to show theoperability of the claimed invention. Those in the art will recognizethat this example indicates the utility of the invention for both invitro and in vivo splicing reactions. While significant utility will beattained in vivo by use of the present invention, those in the art willalso recognize that in vitro utility is important and can be used tocreate chimeric transcripts for use in laboratory situations or in aclinical setting.

EXAMPLE 1 LacZ Fusion

To assess the feasibility of the targeted trans-splicing approach, wetested the ability of the Tetrahymena ribozyme to correct truncated LacZtranscripts with targeted trans-splicing. It has previously been shownthat in E. coli the Tetrahymena self-splicing group I intron canefficiently splice itself from transcripts encoding the alpha-complementof β-galactosidase (β-gal) (FIG. 2A) (J. V. Price, T. R. Cech, Science228, 719 (1985); Waring et al., Cell 40, 371 (1985)). Since thisreaction proceeded very efficiently in cis, we decided to determine ifthe ribozyme could perform a similar reaction in trans.

This system consists of 2 RNA molecules (FIG. 2B): a ribozyme-3′ exonRNA and a 5′ exon RNA. The group I ribozyme used in this study lacks thefirst 21 nucleotides present in the full length intron from which it isderived (A. J. Zaug, T. R. Cech, Science 231, 470 (1986)). The first 23nucleotides of the 3′ exon are derived from the pre-rRNA 3′ exonsequence from Tetrahymena (M. D. Been, T. R. Cech, Cell 47, 207 (1986)).This 23 nucleotide sequence is fused in-frame to 200 nucleotides of thealpha-complement of the LacZ gene (Been and Cech, supra). The 39nucleotide 5′ exon contains a ribosome binding site, the first 21 codingnucleotides of an alpha-complement LacZ transcript, the ribozymerecognition sequence CCCUCU, and two adenosines. These adenosines mustbe removed if trans-splicing is to correct these LacZ transcripts. (FIG.2B). [Previous studies have shown that the sequence and length of theRNA following the CCCUCU is not critical for Tetrahymena ribozyme action(A. J. Zaug, M. D. Been, T. C. Cech, Nature 324, 429 (1986))].

In vitro, the ribozyme can quickly and accurately trans-splice this LacZ3′ exon onto the truncated 39 nucleotide LacZ 5′ exon to generate an RNAproduct which encodes the alpha-complement of β-galactosidase (FIG. 3).The reaction proceeds with speed and efficiency similar to those seen ina reaction with a short 13 nucleotide substrate. The t_(1/2) for thetrans-splicing reaction with the 39 nucleotide substrate was determinedto be 13 minutes under conditions of substrate excess (FIG. 4).

In these experiments, trans-splicing (production of 5′-3′ or 5′L-3′)occurred faster than hydrolysis (production of free 3′ exon; see FIG.2). The rate of hydrolysis of the 3′ exon from the ribozyme wasdetermined to be t_(1/2)−60 minutes in a separate experiment (FIG. 5).An inactive version of the L-21 ribozyme (L-21 del) was not able toperform either the trans-splicing or the hydrolysis reaction (FIGS. 3and 5). Sequencing of the trans-splicing product confirmed that theultimate and penultimate 3′ adenosine nucleotide were correctly removedfrom the 5′ exon-substrate RNA, and this cleaved 5′ exon was accuratelyspliced onto the 3′ exon (data not shown). The splice junction gave theproper reading frame for β-gal expression.

EXAMPLE 2 mRNA Splicing

To determine if targeted trans-splicing could be employed to correctmRNA-size RNA fragments, a transcript which contained the first 1106nucleotides of the LacZ coding sequence as well as signals for in vitrotranslation was created and targeted for alteration by trans-splicing.The L-21 ribozyme was directed to cleave the truncated LacZ transcript19 nucleotides from its 3′ end and trans-splice a 3′ exon brought in bythe ribozyme onto the cleaved LacZ target RNA (FIG. 6A). The 3′ exonsequence attached to the ribozyme encoded the last 1987 nucleotides ofthe LacZ coding sequence and no sequences from the Tetrahymena pre rRNA.Accurate trans-splicing of the 3′ exon sequences onto the truncatedtranscript resulted in a 3074 nucleotide product which encoded theentire LacZ coding sequence (FIG. 6B).

Once again the inactive version of the ribozyme (L-21 del) was unable toperform this reaction, confirming its expected dependence of thecatalytic activity of the RNA itself. The trans-splicing products fromthe 120 minute time points of the reactions shown in FIG. 6 were invitro translated in wheat germ extract, and the in vitro translatedproteins were assayed for β-gal activity using a standard ONPG assay (C.Smith et al., Leukemia 7, 310 (1993)).

Proteins from trans-splicing reactions containing active ribozymes wereshown to contain 1500 units [1000×OD420/(ml-min)] of β-gal activity,while no activity was found in proteins translated from reactionscontaining the inactive ribozyme. Therefore, trans-splicing can beemployed to correct the coding sequence of large defective transcripts.

In the reaction shown in FIG. 6, the labeled substrate RNA is in a 10fold excess to the ribozyme-3′ exon RNA. Therefore, only 10% of thelabeled substrate RNAs could at best be converted to trans-splicedproducts. In this reaction however, we roughly estimate (by comparingdifferent X-ray film exposures of the gel) that at most 1% of thetruncated RNAs are corrected. This lack of efficiency is probably aresult of the targeted RNAs adopting conformations which inhibit theribozyme from correctly interacting with them. To improve the efficiencyof this trans-splicing reaction, alternative sites for cleavage andsplicing which are more accessible to the ribosome can be targeted bystandard manipulation of this experiment. In vivo, cellular proteins mayimprove the efficiency of formation of the correct RNA interaction (Z.Tsuchihashi, M. Khosla, D. Herschlag, Science 262, 99 (1993)).

Uses

Gene mapping and human genome sequencing provides the genetic basis foran increasing number of inherited diseases. With each discovery oridentification of a new disease-related gene there is an opportunity todevelop gene therapy based treatments. Conventional gene therapyapproaches attempt to correct a genetic deficiency by transferring awild-type cDNA copy of a gene under the control of a heterologouspromoter to cells harboring a defective copy of the gene. One obstaclefor implementing such treatments is an inability to faithfullyrecapitulate the normal expression pattern of endogenous genes aftergene transfer (R. A. Morgan, W. F. Anderson, Ann. Rev. Biochem. 62, 191(1993); B. A. Dzierzak, T. Papayannopoulou, R. C. Mulligan, Nature 331,35 (1989)). This may limit the number of genetic diseases treatable bygene therapy. Targeted trans-splicing offers a solution to this problem.

Ribozymes can be used to correct the defective transcripts issuing frommutant genes. This approach will be valuable for the treatment of themany genetic diseases caused by a common set of specific mutations whichdo not affect the expression of the mutant gene. For example, thegenetic basis of many globin diseases is well understood. However, genetherapy based treatments for such diseases have been slow in coming,perhaps, because the expression patterns of the globin genes cannot berecapitulated after gene transfer. Targeted trans-splicing canpotentially repair or correct globin transcripts that are eithertruncated or contain point mutations. In the process, the cellularexpression pattern of these genes is maintained (FIG. 7). Therefore,targeted trans-splicing represents an important, novel strategy for thetreatment of many genetic diseases.

Trans-splicing ribozymes based on any of the self-splicing group Iintrons can be designed to cleave a targeted transcript upstream of aspecific mutation or upstream of a premature 3′ end at essentially anyuridine residue (F. L. Murphy, T. R. Cech, Proc. Natl. Acad. Sci, USA86, 9218 (1989)). One simply changes the sequence of the internal guidesequence within the ribozyme (5′-GNNNNN) to match the sequence precedingthe site of target RNA cleavage (5′-N′N′N′N′N′U), where N-N′ representany allowable base pair. The 3′ exons attached to the ends of theseribozymes are comprised of a sequence designed to correct the mutanttranscripts being targeted. The ribozyme will both cleave the mutanttranscript and replace the mutant 3′ region by a functional sequence.There is very little sequence requirement for a 3′ axon in thesereactions, so virtually any sequence can serve (J. V. Price, T. R. Cech,Genes and Development 2, 1439 (1988)). Thus, trans-splicing ribozymescan be made to correct essentially any mutant transcript becausesequence requirements for 5′ cleavage sites and 3′ exons are minimal.

Trans-splicing ribozymes are also be effective antiviral agents. Severalgroups have employed trans-cleaving ribozymes to inhibit viralreplication. Use of such ribozymes results in the destruction of thetargeted viral RNA inside cells (N. Sarver et al., Science 247, 1222(1990)). Thus, the effectiveness of these trans-cleavage ribozymes restsupon their ability to destroy the vast majority of the targeted viralRNAs. We propose employing trans-splicing ribozymes not to destroy viralRNAs, but to change the sequence of the viral RNAs to give themantiviral activity. For example, the HIV transcripts that encode the gagprotein can be changed to encode a dominant negative version of thisprotein via targeted trans-splicing (FIG. 8) (M. H. Malim, E. Bohniein,J. Hauber, B. R. Culien, Cell 58, 205 (1989); D. Trono, M. B. Feinberg,D. Baltimore, Cell 59, 113 (1989)) or to contain a large number of TARor RRE decoy RNAs (B. A. Sullenger, H. F. Gallardo, G. E. Ungers, E.Gilboa, Cell 63, 601 (1990)).

In contrast to trans-cleaving ribozymes, such antiviral trans-splicingribozymes would have to affect only a small percentage of the targetedHIV transcripts to be effective at inhibiting viral replication. Ingeneral, the ability to change the information encoded by targetedtranscripts by trans-splicing represents a broad new approach to geneinhibition because now transcripts can be altered to encode proteins orRNAs which can inhibit the function of the targeted gene. In otherwords, with targeted trans-splicing, deleterious transcripts can beturned against themselves.

As noted above, trans-splicing may also be accomplished without the useof ribozymes. It has been demonstrated that spliced leader sequencesfrom lower eucaryotes can be trans-spliced onto mammalian 3′ splicesites in tissue culture cells (J. P. Bruzik, T. Maniatis, Nature 360,692 (1992)). Trans-splicing in this case is mediated by the spliceosomeor splicing factors. Thus, it is possible to employ spliceosomes toalter the sequence of targeted transcripts for some desired end viatargeted trans-splicing.

Thus, this invention provides a means for performing molecularreconstructive surgery. A defective part of a useful RNA molecule can becut away from the rest of the molecule and subsequently replaced by afunctional part. Alternatively, a functional portion of adisease-causing or deleterious RNA can be replaced by an inhibitoryportion.

Administration

The above trans-splicing factors or agents can be administered bystandard techniques, some of which are discussed below. They may beadministered as RNA or expressed from expression vectors. Selectedagents, e.g., oligonucleotides or ribozymes can be administeredprophylactically, or to patients suffering from a target disease, e.g.,by exogenous delivery of the agent to an infected tissue by means of anappropriate delivery vehicle, e.g., a liposome, a controlled releasevehicle, by use of iontophoresis, electroporation or ion pairedmolecules, or covalently attached adducts, and other pharmacologicallyapproved methods of delivery. Routes of administration includeintramuscular, aerosol, oral (tablet or pill form), topical, systemic,ocular, intraperitoneal and/or intrathecal. Expression vectors forimmunization with ribozymes and/or delivery of oligonucleotides are alsosuitable.

The specific delivery route of any selected agent will depend on the useof the agent. Generally, a specific delivery program for each agent willfocus on naked agent uptake with regard to intracellular localization,followed by demonstration of efficacy. Alternatively, delivery to thesesame cells in an organ or tissue of an animal can be pursued. Uptakestudies will include uptake assays to evaluate, e.g., cellularoligonucleotide uptake, regardless of the delivery vehicle or strategy.Such assays will also determine the intracellular localization of theagent following uptake, ultimately establishing the requirements formaintenance of steady-state concentrations within the cellularcompartment containing the target sequence (nucleus and/or cytoplasm).Efficacy and cytotoxicity can then be tested. Toxicity will not onlyinclude cell viability but also cell function.

Some methods of delivery that may be used include:

-   -   a. encapsulation in liposomes,    -   b. transduction by retroviral vectors,    -   c. conjugation with cholesterol,    -   d. localization to nuclear compartment utilizing antigen binding        site found on most snRNAs,    -   e. neutralization of charge of ribozyme by using nucleotide        derivatives, and    -   f. use of blood stem cells to distribute ribozymes throughout        the body.

At least three types of delivery strategies are useful in the presentinvention, including: ribozyme modifications, particle carrier drugdelivery vehicles, and retroviral expression vectors. Unmodifiedribozymes and antisense oligonucleotides, like most small molecules, aretaken up by cells, albeit slowly. To enhance cellular uptake, theribozyme may be modified essentially at random, in ways which reduce itscharge but maintain specific functional groups required for RNA cleavageand splicing activity. This results in a molecule which is able todiffuse across the cell membrane, thus removing the permeabilitybarrier.

Modification of ribozymes to reduce charge is just one approach toenhance the cellular uptake of these larger molecules. The randomapproach, however, is not advisable since ribozymes are structurally andfunctionally more complex than small drug molecules. The structuralrequirements necessary to maintain ribozyme catalytic activity are wellunderstood by those in the art. (See, Cech, Curr. Op. Structural Biol.,1992). These requirements are taken into consideration when designingmodifications to enhance cellular delivery. The modifications are alsodesigned to reduce susceptibility to nuclease degradation. Both of thesecharacteristics should greatly improve the efficacy of the ribozyme.Cellular uptake can be increased by several orders of magnitude withouthaving to alter the phosphodiester linkages necessary for ribozymecleavage activity.

Chemical modifications of the phosphate backbone will reduce thenegative charge thereby facilitating diffusion across the membrane. Thisprinciple has been successfully demonstrated for antisense DNAtechnology. The similarities in chemical composition between DNA and RNAmake this a feasible approach. In the body, maintenance of an externalconcentration will be necessary to drive the diffusion of the modifiedribozyme into the cells of the tissue. Administration routes which allowthe diseased tissue to be exposed to a transient high concentration ofthe drug, which is slowly dissipated by systemic adsorption arepreferred. Intravenous administration with a drug carrier designed toincrease the circulation half-life of the ribozyme can be used. The sizeand composition of the drug carrier restricts rapid clearance from theblood stream. The carrier, made to accumulate at the site of infection,can protect the ribozyme from degradative processes.

Drug delivery vehicles are effective for both systemic and topicaladministration. They can be designed to serve as a slow releasereservoir, or to deliver their contents directly to the target cell. Anadvantage of using direct delivery drug vehicles is that multiplemolecules are delivered per uptake. Such vehicles have been shown toincrease the circulation half-life of drugs which would otherwise berapidly cleared from the blood stream. Some examples of such specializeddrug delivery vehicles which fall into this category are liposomes,hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesivemicrospheres.

From this category of delivery systems, liposomes are preferred.Liposomes increase intracellular stability, increase uptake efficiencyand improve biological activity. Liposomes are hollow spherical vesiclescomposed of lipids arranged in a similar fashion as those lipids whichmake up the cell membrane. They have an internal aqueous space forentrapping water soluble compounds and range in size from 0.05 toseveral microns in diameter. Several studies have shown that liposomescan deliver RNA to cells and that the RNA remains biologically active.

For example, a liposome delivery vehicle originally designed as aresearch tool, Lipofectin, has been shown to deliver intact mRNAmolecules to cells yielding production of the corresponding protein.

Liposomes offer several advantages: They are non-toxic and biodegradablein composition; they display long circulation half-lives; andrecognition molecules can be readily attached to their surface fortargeting to tissues. Finally, cost effective manufacture ofliposome-based pharmaceuticals, either in a liquid suspension orlyophilized product, has demonstrated the viability of this technologyas an acceptable drug delivery system.

Other controlled release drug delivery systems, such as nonoparticlesand hydrogels may be potential delivery vehicles for a ribozyme. Thesecarriers have been developed for chemotherapeutic agents andprotein-based pharmaceuticals, and consequently, can be adapted forribozyme delivery.

Topical administration of trans-splicing ribozymes is advantageous sinceit allows localized concentration at the site of administration withminimal systemic adsorption. This simplifies the delivery strategy ofthe ribozyme to the disease site and reduces the extent of toxicologicalcharacterization. Furthermore, the amount of material to be applied isfair less than that required for other administration routes. Effectivedelivery requires the ribozyme to diffuse into the infected cells.Chemical modification of the ribozyme to neutralize negative charge maybe all that is required for penetration. However, in the event thatcharge neutralization is insufficient, the modified ribozyme can beco-formulated with permeability enhancers, such as Azone or oleic acid,in a liposome. The liposomes can either represent a slow releasepresentation vehicle in which the modified ribozyme and permeabilityenhancer transfer from the liposome into the infected cell, or theliposome phospholipids can participate directly with the modifiedribozyme and permeability enhancer in facilitating cellular delivery. Insome cases, both the ribozyme and permeability enhancer can beformulated into a suppository formulation for slow release.

Such ribozymes may also be systemically administered. Systemicabsorption refers to the accumulation of drugs in the blood streamfollowed by distribution throughout the entire body. Administrationroutes which lead to systemic absorption include: intravenous,subcutaneous, intraperitoneal, intranasal, intrathecal and ophthalmic.Each of these administration routes expose the ribozyme to an accessiblediseased tissue. Subcutaneous administration drains into a localizedlymph node which proceeds through the lymphatic network into thecirculation. The rate of entry into the circulation has been shown to bea function of molecular weight or size. The use of a liposome or otherdrug carrier localizes the ribozyme at the lymph node. The ribozyme canbe modified to diffuse into the cell, or the liposome can directlyparticipate in the delivery of either the unmodified or modifiedribozyme to the cell.

A liposome formulation which can associate ribozymes with the surface oflymphocytes and macrophages is also useful. This will provide enhanceddelivery to HIV-infected cells by taking advantage of the specificity ofmacrophage and lymphocyte immune recognition of infected cells. Wholeblood studies show that the formulation is taken up by 90% of thelymphocytes after 8 hours at 37° C. Preliminary biodistribution andpharmacokinetic studies yielded 70% of the injected dose/gm of tissue inthe spleen after one hour following intravenous administration.

Intraperitoneal administration also leads to entry into the circulationwith the molecular weight or size of the ribozyme-delivery vehiclecomplex controlling the rate of entry.

Liposomes injected intravenously show accumulation in the liver, lungand spleen. The composition and size can be adjusted so that thisaccumulation represents 30% to 40% of the injected dose. The rest isleft to circulate in the blood stream for up to 24 hours.

The chosen method of delivery will result in cytoplasmic accumulation inthe afflicted cells and molecules should have some nuclease-resistancefor optimal dosing. Nuclear delivery may be used but is less preferable.Most preferred delivery methods include liposomes (10-400 nm),hydrogels, controlled-release polymers, microinjection orelectroporation (for ex vivo treatments) and other pharmaceuticallyapplicable vehicles. The dosage will depend upon the disease indicationand the route of administration but should be between 100-200 mg/kg ofbody weight/day. The duration of treatment will extend through thecourse of the disease symptoms, usually at least 14-16 days and possiblycontinuously. Multiple daily doses are anticipated for topicalapplications, ocular applications and vaginal applications. The numberof doses will depend upon disease delivery vehicle and efficacy datafrom clinical trials.

Establishment of therapeutic levels of ribozyme within the cell isdependent upon the rate of uptake and degradation. Decreasing the degreeof degradation will prolong the intracellular half-life of the ribozyme.Thus, chemically modified ribozymes, e.g., with modification of thephosphate backbone, or capping of the 5′ and 3′ ends of the ribozymewith nucleotide analogs may require different dosaging. Descriptions ofuseful systems are provided in the art cited above, all of which ishereby incorporated by reference herein.

Particular diseases that may be treated in this manner include anydisease which can be treated by such RNAs, for example, HSV, HBV, EBV,and HIV infection; as well as various carriers (where the targetmolecule is located in a known cellular compartment).

Any disease caused by a specific set of mutations in a given genes RNAis potentially treatable by using target trans-splicing to correct suchdefective RNAs. Such diseases would include:

-   -   A. β-globin diseases (such as sickle cell anemia), cystic        fibrosis, as well as any other genetic diseases caused by a        point mutations or deletions in RNA.    -   B. Cancers caused by specific mutant oncogene encoding RNAs        (e.g. bcr-abl mRNAs, mutant p53 mRNAs).    -   C. Genetic diseases caused by unstable trinucleotide repeats in        RNAs (e.g. Huntington's disease, fragile X syndrome).

Other embodiments are within the following claims.

1. Method for splicing a target RNA molecule comprising a mutantbeta-globin nucleotide sequence within a cell in culture with a separateRNA molecule comprising a wild type beta-globin nucleotide sequence,wherein a protein product of the target RNA molecule is deleterious tothe cell in which it is located, and wherein the separate RNA moleculeis adapted to form a target RNA molecule with the wild type beta-globinnucleotide sequence in place of mutant beta-globin nucleotide sequencewhen spliced with at least a part of the target RNA molecule, the methodcomprising: contacting the target RNA molecule with a catalytic RNAmolecule comprising the separate RNA molecule, under conditions in whichat least a portion of the separate RNA molecule is spliced with at leasta portion of the target RNA molecule to form the target RNA moleculewith the wild type beta-globin nucleotide sequence in place of mutantbeta-globin nucleotide sequence when spliced with at least a part of thetarget RNA molecule.
 2. The method of claim 1, wherein the catalytic RNAmolecule is active to cleave the target RNA molecule comprising a mutantbeta-globin nucleotide sequence and to splice the separate RNA moleculewith the target RNA molecule comprising a mutant beta-globin nucleotidesequence.
 3. The method of claim 1, wherein the contacting is in vitro.4. The method of claim 3, wherein the contacting comprises providing avector encoding the catalytic RNA molecule, wherein the catalytic RNAmolecule includes the separate RNA molecule comprising a wild-typebeta-globin nucleotide sequence.
 5. The method of claim 1, wherein thecatalytic RNA molecule is derived from a group I or group II intronmolecule.